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Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

Koenig U, Fobker M, Lengauer B, Brandstetter M, Resch GP, Gröger M, Plenz G, Pammer J, Barresi C, Hartmann C, Rossiter H - Autophagy (2015)

Bottom Line: We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells.The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells.Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

View Article: PubMed Central - PubMed

Affiliation: a Research Division of Biology and Pathobiology of the Skin; Department of Dermatology ; Medical University of Vienna ; Vienna , Austria.

ABSTRACT
The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

No MeSH data available.


Related in: MedlinePlus

Characterization of autophagy-deficient atg7−/− HaGls. (A) KRT14 expression in myoepithelial cells of the HaGl visualized by immunohistochemistry against KRT14 in young mice (3-wk-old). These basal layer cells in the ducts are formed by myoepithelial cells, which are mitotically active. Ductal cells with large and round nuclei form the inner surface of the ductal lumen. The lumen is partially filled with an amorphous substance from these secretory cells. Myoepithelial cell (→), secretory cell (*), ductal lumen (L). Size bar = 20 μm. (B) Genotyping PCR showing loss of floxed allele in HaGls of Atg7f/fKrt14-cre (atg7−/−) mice. (C) Immunoblot for ATG7, LC3 and GAPDH on Atg7f/f and atg7−/− HaGl tissue homogenates showing almost complete absence of ATG7 (upper band, lower band represents unspecific signal) and of the processed LC3-II form of LC3-I. The lack of the LC3-II band in atg7−/− samples demonstrates abrogation of autophagy. Note: The presence of residual ATG7 protein likely results from other cell types present in the HaGl, such as blood cells. (D) Sections of GFP-LC3 transgenic Atg7f/f and atg7−/− mice showing the presence of a discrete number of ductal cells that display GFP puncta reminiscent of autophagosomes in Atg7f/f. Note: The cell in the center of the Atg7f/f section is binucleated, a feature of the HaGl. In contrast, and in agreement with the increase in LC3-I in the atg7−/− background, GFP-LC3 is diffusely distributed and accumulates in these samples, indicative of free form of GFP-LC3 not incorporated into autophagic vesicles. Note: a few GFP spots can still be detected in atg7−/− cells possibly resulting from GFP aggregates. *, vacuoles completely excluding GFP; arrow, small spots also excluding GFP found in both genotypes; L, ductal lumen. Nuclei were visualized with Hoechst. Size bar = 10 μm.
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f0001: Characterization of autophagy-deficient atg7−/− HaGls. (A) KRT14 expression in myoepithelial cells of the HaGl visualized by immunohistochemistry against KRT14 in young mice (3-wk-old). These basal layer cells in the ducts are formed by myoepithelial cells, which are mitotically active. Ductal cells with large and round nuclei form the inner surface of the ductal lumen. The lumen is partially filled with an amorphous substance from these secretory cells. Myoepithelial cell (→), secretory cell (*), ductal lumen (L). Size bar = 20 μm. (B) Genotyping PCR showing loss of floxed allele in HaGls of Atg7f/fKrt14-cre (atg7−/−) mice. (C) Immunoblot for ATG7, LC3 and GAPDH on Atg7f/f and atg7−/− HaGl tissue homogenates showing almost complete absence of ATG7 (upper band, lower band represents unspecific signal) and of the processed LC3-II form of LC3-I. The lack of the LC3-II band in atg7−/− samples demonstrates abrogation of autophagy. Note: The presence of residual ATG7 protein likely results from other cell types present in the HaGl, such as blood cells. (D) Sections of GFP-LC3 transgenic Atg7f/f and atg7−/− mice showing the presence of a discrete number of ductal cells that display GFP puncta reminiscent of autophagosomes in Atg7f/f. Note: The cell in the center of the Atg7f/f section is binucleated, a feature of the HaGl. In contrast, and in agreement with the increase in LC3-I in the atg7−/− background, GFP-LC3 is diffusely distributed and accumulates in these samples, indicative of free form of GFP-LC3 not incorporated into autophagic vesicles. Note: a few GFP spots can still be detected in atg7−/− cells possibly resulting from GFP aggregates. *, vacuoles completely excluding GFP; arrow, small spots also excluding GFP found in both genotypes; L, ductal lumen. Nuclei were visualized with Hoechst. Size bar = 10 μm.

Mentions: The Harderian gland is an epithelial derived organ with a layer of luminal columnar epithelial cells surrounded by basal myoepithelial cells.29 In young mice myoepithelial cells express smooth muscle differentiation markers in addition to those of basal keratinocytes and are progenitors of luminal cells. In adulthood myoepithelial cells differentiate to myofibroblasts thereby loosing basal epithelial markers. Thus we investigated the expression of the epithelial KRT14 (keratin 14) in the Harderian gland of young mice. As seen in the immunohistochemistry of 3-wk-old mice, basal myoepithelial cells, morphologically characterized by an elongated nucleus parallel to the basement membrane, stain positive for KRT14 (Fig. 1A). Hence a transgenic line expressing Cre recombinase under control of a Krt14 promoter can be used as a deletion-trigger in the Harderian gland.Figure 1.


Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

Koenig U, Fobker M, Lengauer B, Brandstetter M, Resch GP, Gröger M, Plenz G, Pammer J, Barresi C, Hartmann C, Rossiter H - Autophagy (2015)

Characterization of autophagy-deficient atg7−/− HaGls. (A) KRT14 expression in myoepithelial cells of the HaGl visualized by immunohistochemistry against KRT14 in young mice (3-wk-old). These basal layer cells in the ducts are formed by myoepithelial cells, which are mitotically active. Ductal cells with large and round nuclei form the inner surface of the ductal lumen. The lumen is partially filled with an amorphous substance from these secretory cells. Myoepithelial cell (→), secretory cell (*), ductal lumen (L). Size bar = 20 μm. (B) Genotyping PCR showing loss of floxed allele in HaGls of Atg7f/fKrt14-cre (atg7−/−) mice. (C) Immunoblot for ATG7, LC3 and GAPDH on Atg7f/f and atg7−/− HaGl tissue homogenates showing almost complete absence of ATG7 (upper band, lower band represents unspecific signal) and of the processed LC3-II form of LC3-I. The lack of the LC3-II band in atg7−/− samples demonstrates abrogation of autophagy. Note: The presence of residual ATG7 protein likely results from other cell types present in the HaGl, such as blood cells. (D) Sections of GFP-LC3 transgenic Atg7f/f and atg7−/− mice showing the presence of a discrete number of ductal cells that display GFP puncta reminiscent of autophagosomes in Atg7f/f. Note: The cell in the center of the Atg7f/f section is binucleated, a feature of the HaGl. In contrast, and in agreement with the increase in LC3-I in the atg7−/− background, GFP-LC3 is diffusely distributed and accumulates in these samples, indicative of free form of GFP-LC3 not incorporated into autophagic vesicles. Note: a few GFP spots can still be detected in atg7−/− cells possibly resulting from GFP aggregates. *, vacuoles completely excluding GFP; arrow, small spots also excluding GFP found in both genotypes; L, ductal lumen. Nuclei were visualized with Hoechst. Size bar = 10 μm.
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Related In: Results  -  Collection

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f0001: Characterization of autophagy-deficient atg7−/− HaGls. (A) KRT14 expression in myoepithelial cells of the HaGl visualized by immunohistochemistry against KRT14 in young mice (3-wk-old). These basal layer cells in the ducts are formed by myoepithelial cells, which are mitotically active. Ductal cells with large and round nuclei form the inner surface of the ductal lumen. The lumen is partially filled with an amorphous substance from these secretory cells. Myoepithelial cell (→), secretory cell (*), ductal lumen (L). Size bar = 20 μm. (B) Genotyping PCR showing loss of floxed allele in HaGls of Atg7f/fKrt14-cre (atg7−/−) mice. (C) Immunoblot for ATG7, LC3 and GAPDH on Atg7f/f and atg7−/− HaGl tissue homogenates showing almost complete absence of ATG7 (upper band, lower band represents unspecific signal) and of the processed LC3-II form of LC3-I. The lack of the LC3-II band in atg7−/− samples demonstrates abrogation of autophagy. Note: The presence of residual ATG7 protein likely results from other cell types present in the HaGl, such as blood cells. (D) Sections of GFP-LC3 transgenic Atg7f/f and atg7−/− mice showing the presence of a discrete number of ductal cells that display GFP puncta reminiscent of autophagosomes in Atg7f/f. Note: The cell in the center of the Atg7f/f section is binucleated, a feature of the HaGl. In contrast, and in agreement with the increase in LC3-I in the atg7−/− background, GFP-LC3 is diffusely distributed and accumulates in these samples, indicative of free form of GFP-LC3 not incorporated into autophagic vesicles. Note: a few GFP spots can still be detected in atg7−/− cells possibly resulting from GFP aggregates. *, vacuoles completely excluding GFP; arrow, small spots also excluding GFP found in both genotypes; L, ductal lumen. Nuclei were visualized with Hoechst. Size bar = 10 μm.
Mentions: The Harderian gland is an epithelial derived organ with a layer of luminal columnar epithelial cells surrounded by basal myoepithelial cells.29 In young mice myoepithelial cells express smooth muscle differentiation markers in addition to those of basal keratinocytes and are progenitors of luminal cells. In adulthood myoepithelial cells differentiate to myofibroblasts thereby loosing basal epithelial markers. Thus we investigated the expression of the epithelial KRT14 (keratin 14) in the Harderian gland of young mice. As seen in the immunohistochemistry of 3-wk-old mice, basal myoepithelial cells, morphologically characterized by an elongated nucleus parallel to the basement membrane, stain positive for KRT14 (Fig. 1A). Hence a transgenic line expressing Cre recombinase under control of a Krt14 promoter can be used as a deletion-trigger in the Harderian gland.Figure 1.

Bottom Line: We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells.The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells.Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

View Article: PubMed Central - PubMed

Affiliation: a Research Division of Biology and Pathobiology of the Skin; Department of Dermatology ; Medical University of Vienna ; Vienna , Austria.

ABSTRACT
The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

No MeSH data available.


Related in: MedlinePlus